Effective γδ T‐cell clinical therapies: current limitations and future perspectives for cancer immunotherapy

Abstract γδ T cells are a unique subset of T lymphocytes, exhibiting features of both innate and adaptive immune cells and are involved with cancer immunosurveillance. They present an attractive alternative to conventional T cell‐based immunotherapy due, in large part, to their lack of major histocompatibility (MHC) restriction and ability to secrete high levels of cytokines with well‐known anti‐tumour functions. To date, clinical trials using γδ T cell‐based immunotherapy for a range of haematological and solid cancers have yielded limited success compared with in vitro studies. This inability to translate the efficacy of γδ T‐cell therapies from preclinical to clinical trials is attributed to a combination of several factors, e.g. γδ T‐cell agonists that are commonly used to stimulate populations of these cells have limited cellular uptake yet rely on intracellular mechanisms; administered γδ T cells display low levels of tumour‐infiltration; and there is a gap in the understanding of γδ T‐cell inhibitory receptors. This review explores the discrepancy between γδ T‐cell clinical and preclinical performance and offers viable avenues to overcome these obstacles. Using more direct γδ T‐cell agonists, encapsulating these agonists into lipid nanocarriers to improve their pharmacokinetic and pharmacodynamic profiles and the use of combination therapies to overcome checkpoint inhibition and T‐cell exhaustion are ways to bridge the gap between preclinical and clinical success. Given the ability to overcome these limitations, the development of a more targeted γδ T‐cell agonist‐checkpoint blockade combination therapy has the potential for success in clinical trials which has to date remained elusive.


INTRODUCTION
In an aging population, where cancer diagnoses are increasingly prevalent, the pursuit of effective treatments remains a high priority area for biomedical research.Current cancer treatments are typically various combinations of chemotherapy, surgery or radiotherapy; however, these treatments all present challenges with off-target effects and relapse that can render treatment less effective. 1To date, immunotherapy presents one of the more clinically successful forms of treatment and is an ever-expanding area of research and development.Checkpoint inhibitors (such as programmed cell death protein 1 (PD-1) or Cytotoxic T-Lymphocyte Associated protein 4 (CTLA-4) inhibitors) present one avenue of immunotherapy that aim to circumvent the immunosuppression inherent to many cancers and thus, enable immune cells to carry out their 'anti-tumour' roles. 2 However, checkpoint inhibitors have only proven effective in certain cancer types, and even in these cohorts, just a fraction of patients experience clinical benefit following treatment. 3lternatively, chimeric antigen receptor T cell (CAR T cell)-based therapy has been developed in attempts to improve the immune response against malignant cells. 4CAR T cells are synthesised by transfection of CAR genes into T cells that are taken either from the patient that will be treated (autologous) or from healthy donors (allogenic). 4][6] The limited success with CAR T-cell treatments in solid tumours is attributed to challenges with tumour infiltration of immune cells, heterogeneous expression of tumour-associated antigens, immunosuppressive tumour microenvironments (TMEs) as well as CAR-T-cell-related toxicity. 4,6d T cells are a unique subset of T lymphocytes and offer an attractive option for T cell-based immunotherapy due to their ability to secrete high levels of cytokines with anti-tumour functions while displaying features of both innate and adaptive immune cells. 2,7,8These features, as well as the ability to recognise a broad range of ligands, have resulted in pre-clinical and clinical trials using cd T cells that are well-tolerated and display minimal off-target effects, particularly in comparison to traditional T-cell therapies. 2][11] Phosphoantigens (pAgs) are bacterial or selfderived molecules that can more directly stimulate populations of cd T cells than traditional n-BPs. 2,12,13fortunately, pAgs also have limited use as immunotherapeutic agents due to suboptimal pharmacodynamics and kinetics. 14The development of new therapeutics that have the capacity to overcome systemic toxicity and immune suppression remains a high priority for cancer research.
This review seeks to highlight and account for the discrepancies between cd T-cell immunotherapeutic in vitro studies and clinical trials.cd T-cell therapy has been shown to be well-tolerated and potentially presents a way to overcome immune evasion mechanisms often employed by cancerous cells. 15,16A high level of tumour-infiltrating cd T cells serves as a good indicator of favourable prognostic outcomes, yet thus far, this has not translated into successful clinical trials. 8Improving the efficacy of cd T-cell immunotherapies is a relatively under-explored area of research, yet such approaches hold great promise due to the many favourable features that cd T-cell therapies display in comparison to conventional ab T-cell therapies.Through a better understanding of checkpoint inhibition mechanisms relevant for cd T cells and improved pharmacodynamics of cd T-cell agonists, an 'offthe-shelf' immunotherapeutic treatment for solid and haematological cancers may be within reach.

cd T CELLS
Tumours develop within unique microenvironments which contain a broad spectrum of cells, soluble factors and bacteria, all of which can contribute to the growth and invasiveness of the tumour.The tumour microenvironment (TME) includes soluble factors such as cytokines and chemokines, as well as stromal, endothelial and immune cells that can function to aid tumour cells in proliferating and invading host tissues. 17The immune cells found within the TME are heterogeneous populations, belonging to both the adaptive and innate immune systems, and can elicit either pro or anti-tumour responses. 18Cells of the TME include tumour associated macrophages (TAMs), natural killer cells (NKs) dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs) and T lymphocytes.Tumour progression and prognosis is largely dependent upon the type of infiltrating immune cells as well as the influence of the TME on activation or suppression of these cells. 17,19,20d T cells are a subset of T lymphocytes which constitute 1-10% of T cells in the peripheral blood of healthy adults and contribute to populations of tumour-infiltrating immune cells. 7his subset of T cells expresses T-cell receptors (TCRs) that are composed of c and d chains, making them distinct from T cells expressing TCRs composed of a and b chains. 21,22While a major proportion of the T cells in the body express ab TCRs, cd T cells have garnered interest in recent years due to various features that are unique to this population of lymphocytes that make them ideal targets for cancer immunotherapy. 23cd T cells are unique in that they do not require major histocompatibility complex (MHC) for antigen presentation. 21This is advantageous for a number of reasons but especially in immunotherapy, as the efficacy of many traditional T cell-based immunotherapies is limited by the ability of cancer cells to downregulate MHC class I expression as a means of avoiding immune detection. 24n humans, cd T cells can be categorised according to expression of the d chain of the TCR and display variable expression of the c chain of the TCR.There are three main populations of cd T cells in humans which are classed as Vd1 + , Vd2 + and Vd3 + which reside in differing proportions in various tissues. 18,21,25Through clonal rearrangement of c and d chains, cd T cells can carry out a diverse range of functions in response to various ligands which has been reviewed extensively elsewhere and many of which remain to be identified. 21,23,26In general, the functions of cd T cells are many and varied and this in part accounts for the difficulty researchers have had in elucidating their specific role within the immune system.cd T cells link the innate and adaptive immune systems by detecting altered cellular metabolism and are involved in immune surveillance and responses following infection, tissue damage and cellular transformation. 21ike other subsets of immune cells, the functions carried out by cd T cells are largely dependent upon their polarisation which is ultimately influenced by the microenvironment that the cells are exposed to, which will be discussed in detail further on. 7he nature of ligand recognition by cd T cells remains an area of interest, as the mechanisms involved are not well understood.The difficulty in identifying the molecular mechanisms for the activation of cd T cells is largely owing to the ability of these lymphocytes to recognise such a diverse range of ligands. 21This sits in contrast with conventional ab T cells, which are limited to the recognition of peptide antigens.Broadly, cd T cells are considered to be 'stress sensors' and can detect altered metabolism within both pathogenic and host cells. 26As highlighted in Figure 1, receptors shared by NK cells of the innate immune system are also expressed by cd T cells such as NKG2D, NKp30 and NKp44, aiding in the detection of stress-induced ligands. 7,27hile cd T cells can recognise a wide range of molecules, which are extensively reviewed elsewhere, pAg-induced activation of a particular subset of cd T cells known as Vd2Vc9 T cells is currently the best understood mechanism because of its association with cancer immunotherapy. 7,27,28owever, it is important to note that pAg-induced activation of Vd2Vc9 T cells is not exclusive to cancer and has also been demonstrated in a diverse range of infectious diseases such as tuberculosis, toxoplasmosis and even with SARS-CoV-2. 29Ags are naturally occurring intermediates of the mevalonate and non-mevalonate pathways of isoprenoid biosynthesis and are known to be the main agonists of peripheral blood Vd2Vc9 cd T cells. 21,23The primary source of pAgs in humans is the mevalonate pathway, intermediates of which accumulate intracellularly when this pathway is dysregulated such as occurs in hypercholesterolemia and other cardiovascular diseases, and has implications with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. 21,30,31Vd2Vc9 T cells are uniquely equipped to recognise foreign and tumour-derived pAgs without the need for antigen processing or presentation. 17,21,32Detection of intracellular pAgs by cd T cells relies on an 'inside-out' signalling mechanism involving the family of butyrophilin and butyrophilin-like molecules. 21,23Until recently, it was thought that the transmembrane protein BTN3A1 would bind pAgs and directly present them to Vd2Vc9 TCRs. 33][39] Upon activation by pAgs, Vd2Vc9 T cells tend to express a Th 1 -like phenotype and secrete the proinflammatory cytokines IFN-c and TNF-a. 19he cytolytic activity of Vd2Vc9 T cells can also be increased by stimulation with pAgs in conjunction with interleukin-2 (IL-2) by causing the upregulation of the surface receptor CD16 which upon activation transduces signals that promote Antibody-Dependent Cellular Cytotoxicity (ADCC) in response to specific antibodies. 23These are just some of the ways in which pAg-induced activation of cd T cells can induce a variety of effector functions that make them particularly suited for immunosurveillance and killing of malignant or infected cells.

Role of cd T cells in cancer
The role of cd T cells in cancer progression remains somewhat controversial due to the plasticity that these cells display.cd T cells have been detected in many haematological and solid cancers where in some cases, higher levels of circulating and tumour-infiltrating cd T cells have been associated with better prognosis while in other cases, the opposite has been suggested. 19iven that the primary source of pAgs in humans is via the mevalonate pathway, which is often upregulated in transformed cells, it is not surprising that Vd2Vc9 T cells are involved in cancer immunosurveillance. 21,40Aside from via pAgs, Vd2Vc9 T cells can also identify tumour cells through interaction with tumour-associated surface proteins such as F1-ATPase, NKG2D, TRAIL and CD226 which promote cytolytic activity. 2,21,41d2Vc9 T cells may be attracted to the TME via chemokines where they can detect transformed cells; however, few studies to date have investigated the specific receptors expressed by cd T cells in relation to TME chemokines. 8It is thought that the TME is a major factor in the polarisation of tumourinfiltrating cd T cells to a pro-tumour phenotype, thus limiting their effectiveness as cytotoxic cells. 17,19,21][43][44] The anti-tumour role of cd T cells is relatively well-established due to the wide range of effector functions that they are known to carry out upon activation.cd T cells can act through both direct and indirect pathways to kill transformed cells, which has been demonstrated both in vitro and in vivo in multiple types of cancer. 26Higher levels of circulating and tumour-infiltrating cd T cells have been correlated with a more favourable prognostic outcome across 25 different solid and haematological cancers, including Acute Myeloid Leukaemia (AML), breast cancer, colon cancer and Burkitt's lymphoma. 8,45Cytotoxicity from cd T cells against transformed cells has been suggested to be largely due to dysregulation of the mevalonate pathway, which is often observed in tumours with p53 mutations, leading to the accumulation of endogenous pAgs. 21Another potential source of Vd2Vc9-induced cytotoxicity against cancer cells is hypothesised to be due to the production of pAgs by bacteria within the oncobiome. 23,46PAginduced activation of cd T cells results in the secretion of inflammatory cytokines such as IFN-c and TNF-a which have strong anti-tumour activity, inhibiting angiogenesis as well as inducing DC maturation. 41,47However, pAg-induced activation of Vd2Vc9 T cells may result in different outcomes, depending on the cytokine profile of the TME. 41gure 2. The pro (red) and anti-tumour (green) roles of Vc9Vd2 + T cells and the cytokines involved in differentiation into various effector/ regulatory functions following pAg-induced activation. 101,103This figure is adapted from Imbert and Olive 8 and Pang et al., 105 and was created using Biorender.
In recent years, the pro-tumour role of cd T cells has also been realised, although the full extent that these immune cells play in the promotion of tumorigenesis is not fully understood.It is well known that the TME has an abundance of immunosuppressive cytokines such as TGF-b and IL-10 which can suppress the normal cytotoxic functions of immune cells. 19Figure 2 highlights the polarising effect of these cytokines on cd T cells, which can cause them to express a T reg -like or Th 17 phenotype. 19][50] The secretion of IL-8 and GM-CSF by Th 17 cd T cells has been shown to attract immunosuppressive neutrophils to tumour sites. 2,51While the pro-tumour role of cd T cells is not as well understood as their anti-tumour role, it is nonetheless an important consideration in the efficacy of cd T cell-based immunotherapies.

Inhibitory receptors
Following immune cell activation, there is a need for mechanisms that prevent uncontrolled activation and proliferation in order to avoid damage to healthy tissues and chronic inflammation. 52One such mechanism that immune cells employ is the expression of immune checkpoint receptors. 52,53ecently, 3D melanoma models have been used to demonstrate the limited anti-tumour capacity of tumour-infiltrating cd T cells due to increased expression of checkpoint receptors such as PD-1 and CTLA-4. 54In this study, cd T-cell anti-tumour activity and tumour infiltration were enhanced through the use of checkpoint inhibitors to overcome T-cell exhaustion. 54ome progress has been made in recent years in identifying the checkpoint receptors expressed by cd T cells, some of which are summarised in Figure 3. B and T Lymphocyte Attenuator (BTLA) is an inhibitory receptor expressed by the majority of lymphocytes. 55Herpesvirus Entry Mediator (HVEM) is the natural ligand for BTLA and HVEM positive cells have been shown to inhibit Vc9Vd2 T-cell proliferation through interaction with BTLA. 53,56In fact, BTLA stimulation of Vc9Vd2 T cells may provide a means of immune escape by malignant cells. 53,56BTLA has been shown to be upregulated on the surface of cd T cells in patients with non-small cell lung cancer, further suggesting the involvement of this molecule in immune evasion by malignant cells. 57In a similar way to BTLA, CTLA-4 is a receptor expressed by cd T cells and is involved in regulating their activation. 58To date, CTLA-4 involvement in T-cell checkpoint inhibition has mostly been studied in conventional T cells. 52Recently it was shown that Vd2 + T cells with high levels of CTLA-4 expression displayed suppressed proliferation and cytotoxic potential, further suggesting the involvement of this receptor in cd T-cell exhaustion and suppression. 59D-1 is another inhibitory receptor expressed by cd T cells which likely plays an important role in immune escape by cancerous cells. 60,61In contrast to CTLA-4 and BTLA, PD-1 is more involved with regulating the cytotoxic activity of cd T cells rather than their proliferation. 52,53,57ollowing activation by pAgs, PD-1 has been shown to be upregulated on the surface of cd T cells and PD-1 blockade has resulted in increased IFN-c production by cd T cells as well as enhanced ADCC. 61,62In leukaemic cell lines it was found that in vitro, the combination of pembrolizumaban anti-PD-1 therapyand Zoledronate (ZOL) was only able to significantly increase the production of IFN-c against one out of the four cell lines tested, which was likely a result of variable expression of PD-1 ligand by cancer cell lines. 61Cancer cell lines with high levels of PD-1 Ligand (PD-L1) expression were also tested for the effect of anti-PD-L1 and anti-PD-1 monoclonal antibody treatment on cd T-cell cytotoxicity in vitro. 63It was shown that while anti-PD-1 therapy did not increase cd T-cell cytotoxicity, anti-PD-L1 therapy did increase the cytotoxicity of cd T cells against cancer cells that had been pre-treated with ZOL. 63These contradictory results were shown to be due to cd T-cell ADCC activation occurring with anti-PD-L1 therapy in cancer cell lines that have high levels of PD-L1 expression. 63iller cell immunoglobulin-like receptor 2/3 (KIR2DL2/3) is another inhibitory receptor expressed by cd T cells that has recently been demonstrated to be upregulated upon treatment with the anti-cancer drug decitabine. 64KIR2DL2/3 negative cd T cells displayed enhanced cytotoxicity to tumour cells that expressed the ligand for this inhibitory receptor when compared with KIR2DL2/3 positive cd T cells. 64To date, knowledge about checkpoint inhibitors for cd T cells remains limited but is highly relevant, particularly if immune evasion mechanisms that cancerous cells employ are to be fully understood and overcome in immunotherapy.

CURRENT THERAPIES
There are two main approaches that are utilised for cd T cell-based immunotherapies: in vivo expansion using agonists to activate cells, or the adoptive transfer of ex vivo activated cells.In vivo trials utilising the expansion of cd T cells have thus far shown limited efficacy in terms of cd T-cell tumour infiltration and activation, likely owing to limited knowledge about immunosuppression by the TME on cd T cells, as well as the systemic toxicity displayed by commonly used agonists, such as n-BPs. 26,65Further challenges are presented by the lack of information about immune checkpoint inhibition of cd T cells, which may be involved in inhibiting their efficacy for immunotherapy. 2

Therapeutic cd T-cell agonists
Bisphosphonates are a class of osteoporosis drugs that have been repurposed for cancer treatment due to their ability to expand populations of cd T cells. 9N-BPs were developed to improve the plasma stability of traditional bisphosphonates and target farnesyl pyrophosphate synthase (FPPS)a critical enzyme in the mevalonate pathway, as outlined in Figure 4. 9 Inhibition of FPPS leads to the intracellular accumulation of pAgs, such as isopentenyl pyrophosphate (IPP), which can in turn activate Vd2Vc9 T cells. 21As n-BPs can induce higher levels of endogenous pAgs in phagocytic antigen-presenting cells, this allows for their use as immunotherapeutic agents by initiating Vd2Vc9 T-cell activation. 66N-BPs such as ZOL and alendronate (ALD) are most often used as agonists for the ex vivo expansion of Vd2Vc9 T cells for adoptive cell transfer, as shown in Table 1. 50However, the in vivo use of n-BPs in their unaltered form is limited by their suboptimal pharmacokinetics, pharmacodynamics and biodistribution.When delivered orally, which is the most common route of administration, less than 1% of n-BPs are absorbed. 67,68Even if this intestinal barrier is overcome, n-BPs are small hydrophilic, charged molecules and are rapidly cleared from circulation, either through the renal system or by binding to bone. 66Furthermore, it has been shown that continuous dosing of n-BPs in vivo can induce Vd2Vc9 T-cell exhaustion, thus rendering the treatment ineffective. 50These issues with standard therapeutics that are used to activate cd T cells may in part account for the limited success seen in clinical trials thus far.

Adoptive cd T-cell immunotherapy
To date, cd T-cell adoptive immunotherapies have yielded underwhelming results and any success has been mainly limited to haematological malignancies.The efficacy of adoptive T-cell therapiesparticularly in solid tumoursrelies on the ability of cd T cells to infiltrate tumours, yet even if this challenge is overcome, scalability still presents a major issue. 65In murine models, adoptive immunotherapy using ex vivo Vd2Vc9 T cells expanded by n-BPs has shown efficacy against mesenchymal glioblastomas, 69 osteosarcoma 70 and epithelial ovarian cancer. 71ore recently, adoptive Vd2Vc9 T-cell immunotherapy has shown promising efficacy in clinical settings for late-stage lung and liver cancers 72 as well as non-small cell lung cancer. 73llogenic treatment using cd T cells from healthy donors has proven to be clinically safe and somewhat more efficacious in the treatment of solid tumours than autologous adoptive Vd2Vc9 T-cell immunotherapy, perhaps owing to an ability to overcome the immunosuppressive TME. 72,73cd T cells' lack of MHC restriction  minimises the risk of cross-reactivity and graft vs. host disease in patients. 2,74Allogenic 'off-theshelf' strategies for the treatment of tumours present enormous practical benefit in comparison to autologous therapies which are often complex, time-consuming and costly to produce.A recent clinical trial in late-stage lung or liver cancer patients was performed to establish the safety of allogenic Vd2Vc9 T-cell treatment. 72Patients were injected with 4 9 10 8 Vd2Vc9 T cells that had been cultured from healthy donors, once every 2-3 weeks for the first five infusions, and then either once every month or 2 months after that. 72The results of this study indicated that allogenic Vd2Vc9 T-cell treatment is not only well-tolerated but also efficacious, as almost all patients who received more than five infusions were shown to have significantly prolonged survival. 72AR T-cell therapies are another type of adoptive immunotherapy which typically involves introduction of a CAR transgene into conventional ab T cells.As previously stated, there are many issues facing CAR T-cell therapy using ab T cells and as such, CAR cd T-cell therapies have been the next natural progression in the field.By using cd T cells as the vehicle for CAR T-cell therapies, researchers have been able to combine the specificity of CAR T cells with the advantages of cd T cells, particularly the versatility, scalability and reduced toxicity against healthy tissue.75 The current state of CAR cd T-cell therapies has been extensively reviewed elsewhere but briefly, and further insights into cd T-cell checkpoint mechanisms and interaction with the TME would greatly benefit this field as well.[75][76][77] A major obstacle with adoptive immunotherapies is the biodistribution of T cells following administration, as they must first reach the tumour to successfully target malignant cells.78 In 2011, a phase I clinical trial showed that adoptively transferred autologous cd T cells typically migrate to the lungs, liver and spleen while also being found in some metastatic tumour sites.79 Recently in melanoma xenograft mouse model, tumours that were pre-treated with a liposomal formulation of the n-BP ALD showed decreased accumulation of cd T cells in the liver and increased accumulation at tumour sites following adoptive treatment.78 This was achieved through systemic administration of liposomal n-BPs which then accumulated at tumour sites suggesting the possibility of using passive targeting to direct administered T cells to tumours.78 These promising findings warrant further investigation in different cell lines and cancer models.Given the ability to safely transfer large populations of allogenic Vd2Vc9 T cells and effectively direct transferred cells to tumour sites, adoptive therapy remains a hopeful avenue for cancer treatment.

In vivo cd T-cell immunotherapy
In comparison to adoptive immunotherapy, the in vivo expansion of cd T cells has displayed less success, likely owing to suboptimal delivery of commonly used agonists.][82] A phase I clinical trial in paediatric patients with refractory neuroblastoma who were shown to have significantly decreased counts of cd T cells were treated with ZOL + IL-2 and had on average a 3-to 10-fold increase in circulating cd T cells, restoring levels to a healthy range. 80A 2007 phase I clinical trial using ZOL + IL-2 treatment in patients with hormone refractory prostate cancer showed partial remission in three patients, stable disease in five patients and, interestingly, demonstrated a statistically significant inverse correlation between serum prostate-specific antigen levels and total number of cd T cells after 9 months. 81Another pilot study in patients with advanced haematological malignancies showed on average a 68-fold increase in administered cd T cells after in vivo treatment with ZOL + IL-2. 83A 2012 prospective phase I/II clinical trial in patients with either renal cell carcinoma, malignant melanoma, or AML also showed that treatment with ZOL + IL-2 is well-tolerated and induced partial remission in 25% of AML patients. 82The viability of expanding Vd2Vc9 T-cell populations in vivo has also recently been investigated using an anti-BTN3A agonist which has reached clinical trials. 15nitial results confirmed the treatment is welltolerated and promotes tumour infiltration of immune cells in patients with advanced-stage solid tumours. 15The positive but limited outcomes in these clinical trials with in vivo expansion of cd T cells may be attributed to the small sample sizes as well as these trials being a last chance treatment for advanced stage cancers.An important consideration in these trials is the fact that ZOL + IL-2 treatment is known to induce proliferation of NK cells, hence an antitumour effect from these cells cannot be completely ruled out. 82Given the ability to stimulate populations of cd T cells safely and potently with minimal off-target cytotoxicity, a targeted and efficacious in vivo cancer immunotherapy is within reach.

FUTURE DIRECTIONS FOR CLINICAL TRANSLATION Nanomedicine-based approaches
Nanocarriers have been used in attempts to improve the pharmacodynamic and pharmacokinetic profile of n-BPs for use in cancer patients. 50While many types of nanocarriers have been explored to improve the intracellular delivery of n-BPs, lipid-based nanocarriers have been the most extensively utilised in the clinic due to their well-known biocompatibility and efficacy. 9,21,84ontaining both hydrophobic and hydrophilic regions, as indicated in Figure 5a, lipid-based nanocarriers are capable of encapsulating a wide range of therapeutics and improving their solubility, cell uptake and altering their pharmacokinetic and pharmacodynamic profiles. 84ipid-based nanocarriers have also been shown to preferentially extravasate into and accumulate within tumours due to the leaky vasculature that is typical of many solid cancers, as shown in Figure 5b. 9These nanocarriers also offer a high degree of 'tuneability', allowing for surface modification with targeting ligands and other molecules, and can be used for co-delivery of poorly bioavailable compounds. 1,85Lipid-coated calcium-complexed ZOL nanoparticles have been used to prevent the premature release of ZOL and improve biodistribution to tumours. 86,87In 2012, negatively charged liposomes containing ALD were formulated to target circulating monocytes and macrophages of the mononuclear phagocyte system (MPS). 66These liposomal formulations were shown to stimulate circulating cd T cells via cells of the MPS while simultaneously inhibiting TAMs in a breast cancer mouse model. 66Anti-tumour efficacy that has been observed in mouse models that lack cd T cells has been suggested to be a result of endocytosis of n-BPs by TAMs, as n-BPs have been shown to cause the depletion of these cells. 66,88In hepatocellular carcinoma and triple-negative breast cancer xenograft mouse models, liposomal ZOL was shown to eliminate both M1 anti-tumoral and M2 pro-tumoral TAMs, favouring elimination of M2 macrophages, although this was attributed to the ratio of M1:M2 TAMs in the tumour models used. 89,90Adoptive immunotherapy using Vc9Vd2 T cells activated by liposomal ALD has been demonstrated to be efficacious in the treatment of epithelial ovarian cancer in mice. 91Lipid-based nanocarrier approaches have shown extensive efficacy in the activation of cd T-cell-mediated lysis against cancer cells in vitro and in mouse models yet there are still toxicity and bioavailability issues that must be addressed to achieve clinical efficacy.Leaky vasculature that is common to many solid tumours allows for the enhanced permeability and retention (EPR) effect meaning that small nanoparticles can extravasate from blood vessels into tumours and will not be drained by the lymph system due to impaired lymphatic drainage.This allows nanoparticles to passively accumulate at tumour sites.The figure was created using Biorender.

Next generation cd T-cell agonists
Even with the apparent success of preclinical trials with lipid nanocarrier formulations of n-BPs, there has been limited success in translation to human clinical trials.The lack of human clinical trials that use n-BPs in vivo is most likely due to their systemic cellular toxicity attributed to inhibition of FPPS as well as rapid binding of the phosphate-containing drugs to bone. 2,12While lipid-based nanocarriers have proven efficacious in improving the bioavailability of n-BPs, there remain concerns with low cellular uptake, systemic toxicity and immunosuppression that need to be overcome before n-BPs can be used successfully in the clinic.
As an alternative to n-BPs, pAgs that can potently stimulate populations of cd T cells via more direct mechanisms have been explored for use as immunotherapeutic agents.
(E)-4-Hydroxy-3methyl-but-2-enyl pyrophosphate (HMBPP) is a small pAg molecule that is one of the most potent naturally occurring agonists of Vd2Vc9 T cells.HMBPP is the last intermediate in the bacterial, nonmevalonate pathway of isoprenoid biosynthesis and can accumulate within cells which can be detected by Vd2Vc9 T cells. 12,14,92,93Like other pAgs, HMBPP relies on an 'inside-out' signalling mechanism to activate cd T cells (see Figure 4), and so its use as a therapeutic agent is limited by its inability to passively diffuse across cell membranes. 32The inability of HMBPP to cross cell membranes as well as its instability in plasma means that it has limited ability to potently activate Vd2Vc9 T cells in its naked form, particularly in comparison to charge neutral HMBPP prodrugs that have recently been developed. 94In its natural form, HMBPP requires continuous dosing to achieve biologically relevant concentrations in vivo and for this reason, has had limited success as an immunotherapeutic agent. 93,95harge neutral HMBPP prodrugs, such as BrHPP (Table 1), have been synthesised in attempts to increase cellular uptake, so that this pAg may exert its intracellular effects and stimulate the proliferation of Vd2Vc9 T cells. 14,35,96The aim of a charge neutral HMBPP prodrug is that it may be targeted to malignant cells and more readily cross cell membranes to induce Vd2Vc9 T-cell-mediated killing. 35This method of in vivo Vd2Vc9 T-cell activation relies either on the presence of tumourinfiltrating and accessory leukocytes to aid in strong proliferation of Vd2Vc9 T cells within the TME or on the ability of Vd2Vc9 T cells to infiltrate into tumours, particularly in the case of solid tumours. 32An alternative strategy that has been trialled in vitro is incubating cd T cells with HMBPP to stimulate their proliferation and then co-culturing these cells with cancer cell lines. 19ecently, glioblastoma multiforme cells were shown to induce a Th 1 anti-cancer profile in Vd2Vc9 T cells which had been expanded using HMBPP. 19he in vitro success of HMBPP prodrugs in activating populations of Vd2Vc9 T cells suggests that, given improved in vivo pharmacodynamics and targeting to malignant cells, HMBPP may be a successful immunotherapeutic agent for tumours containing populations of cd T cells.
Recently, a rapid expansion protocol was described for cord blood-derived cd T cells.In this study, lymphoblastoid cells that had been transformed with Epstein-Barr virus were used as feeder cells to stimulate the rapid expansion of cd T cells. 97In comparison to peripheral blood-derived cultures, cord blood-derived cultures showed significantly higher fold expansion of cd T cells which was attributed to the less differentiated nature of cells in cord blood. 98Cord blood cd T cells showed the ability to elicit a broad spectrum of effector functions following expansion, which was not the case for peripheral blood cd T cells which consist largely of the semi-invariant Vc9Vd2 T-cell subtype. 97The results of this study suggest that the unique phenotypic and functional profiles of Vd2Vc9 T cells observed in cord blood may translate to superior cancer cell targeting due to the presence of more diverse effector functions.The scalability of this method as an immunotherapy is likely limited by the accessibility of human cord blood samples.Nevertheless, this study highlights the importance of considering the phenotypic profile of the cell source as it can heavily influence efficacy following expansion.

Combination therapy
Administration or in vivo expansion of cd T cells is often insufficient on its own to overcome many of the immune evasion and suppression mechanisms that malignant cells employ.Treatments that circumvent the mechanisms that cancer cells employ to suppress and evade immune cells present a viable way to improve the efficacy of cd T cell-based immunotherapies.To date, this has proven difficult due to limited knowledge about cd T-cell inhibitory receptors as well as the specific interactions between TME chemokines and cd T cells.Recently, melanoma spheroids were developed for use as a preclinical model to investigate the efficacy of cd T-cell therapy in combination with immune checkpoint inhibitors. 54Treatment of spheroids with cd T cells in combination with anti-CTLA-4 and anti-PD-1 monoclonal antibodies resulted in decreased size of spheroids and displayed increased apoptosis in comparison to treatment with cd T cells alone. 54nother study demonstrated that PD-1 blockade was able to increase the expression of IFN-c in cd T cells, thus enhancing their anti-tumour capacity against AML cells, further suggesting the efficacy of cd T-cell immunotherapy in combination with checkpoint inhibitors. 61n alternative route to enhance the efficacy of cd T-cell immunotherapy has recently been explored that blocks the immunosuppressive interactions of the protein BTN3A1, although the mechanisms through which this occurs are not yet understood.It is suggested that BTN3A1 plays an immunosuppressive role against ab T cells when it is not in a heterodimer conformation with BTN2A1, as has been shown to occur upon pAg-induced activation. 15,36,99Agonistic antibodies targeting BTN3A1 that can mimic the conformational changes that occur upon binding of pAgs have been shown to restore ab T-cell anti-tumour activity while simultaneously inducing cd T-cellmediated cytotoxicity. 36A BTN3A monoclonal antibody has recently reached phase I/IIa clinical trial stage and initial results suggest this treatment is well-tolerated. 15These early results suggest the possibility of cd T-cell-BTN3A1 antibody combination therapy to enhance the cytotoxic activity of administered cd T cells.
The co-administration of cd T cells with activating agents also presents a viable route to improve the anti-tumour capability of administered cells.As evidenced in Table 1, ZOL is often administered in combination with IL-2 due to its ability to cause the proliferation of these cells. 80,100IL-15 secreted by DCs has been shown to enhance the cytotoxicity of cd T cells against haematological cancer cell lines in vitro. 101Clinical trials using IL-15 or IL-15 derivatives have been extensively reviewed elsewhere but generally show that there are still major obstacles to be overcome preventing its use as a monotherapy. 102Interestingly, ascorbic acid (Vitamin C) promotes proliferation of cd T cells and has been shown to increase their production of IFN-c. 103Synergy between vitamin C and anti-PD-1 has also been demonstrated, showing enhanced anti-tumour activity in patients with B cell lymphoma, suggesting the potential benefit of a vitamin C-cd T-cell agonist combination therapy. 104he co-administration of cd T-cell agonists and cytotoxicity-enhancing cytokines and molecules could potentially aid in overcoming exhausted cd T-cell phenotypes, given the ability to avoid the side effects associated with high cytokine levels.

CONCLUSION
Despite the apparent benefits of cd T cells in comparison to conventional T cells, to date, cd T-cell clinical trials have seen limited success.This inability to translate the success seen with cd T-cell immunotherapy in preclinical studies to clinical trials is likely owing to several factors: the suboptimal pharmacodynamics and off-target effects of cd T-cell agonists, a lack of understanding about cd T-cell checkpoint inhibition and tumour suppression mechanisms, as well as the direct polarising effect of the TME cytokines on cd T cells.Furthermore, clinical trials that have been conducted to date, using either in vivo or ex vivo expanded cd T cells, have been tested as a final treatment alternative in patients with advanced stage cancers that have been resistant to other treatments.Given these factors, a lack of efficacy in cd T-cell-based immunotherapeutic clinical trials is not surprising, yet this should not deter from further exploration of this subset of immune cells in the treatment of various cancers.While checkpoint inhibition mechanisms of cd T cells are not fully understood, these cells are not restricted by many of the mechanisms that prevent the efficacy of conventional T-cell immunotherapies.The potential of alternative drug delivery systemssuch as lipid-based nanocarriersto improve the pharmacodynamics of cd T-cell agonists as well as the use of more direct pAgs may present a viable alternative to improve upon currently used approaches.Furthermore, the use of these cd T-cell agonists in combination with checkpoint inhibitors or activating cytokines would likely aid in not only overcoming tumour evasion and suppression mechanisms but in enhancing the cytotoxicity of administered or expanded cd T cells.However, before this becomes a viable option for immunotherapeutic treatment, it is necessary to learn more about the effect of the TME on cd T-cell polarisation and suppression.If further insight about cd T-cell checkpoint inhibitory receptors is gained, it would be possible to further explore combination therapy to develop an 'off-the-shelf' allogenic immunotherapeutic treatment.

Figure 1 .
Figure 1.cd T-cell-mediated cytotoxicity against tumour or infected cells.Inset shows HMBPP binding to the binding pocket of the intracellular B30.2 domain of BTN3A1.HMBPP binding induces conformational changes, allowing for the formation of a BTN3A1/BTN2A1 heterodimer which increases affinity for the Vd2Vc9 T-cell receptor (TCR).This induces the release of anti-inflammatory cytokines such as IFN-c and TNF-a.Natural killer receptors on the surface of Vd2Vc9 T cells can also be activated by stress-induced ligands expressed by target cells, resulting in the release of perforins and granzymes.The figure was created using Biorender, adapted from Mensurado et al.28

Figure 3 .
Figure 3. Inhibitory receptors expressed by cd T cells shown in conjunction with their corresponding ligands.The natural ligand for B and T lymphocyte attenuator (BTLA) is herpesvirus entry mediator (HVEM), CD80/86 expression can inhibit T-cell activation via cytotoxic T-lymphocyte-associated protein 4, programmed cell death protein 1 (PD-1) is inhibited by programmed cell death ligand 1 (PD-L1) and killer cell Immunoglobulin-like receptor (KIR2DL2/3) is associated with human leukocyte antigen I (HLA-I).This figure is adapted from Gao et al., 106 and was created using Biorender.

Figure 4 .
Figure 4. N-BP mechanism of action whereby farnesyl pyrophosphate synthase (FPPS) is inhibited in the mevalonate pathway.FPPS is involved in the conversion of Isopentenyl Pyrophosphate (IPP) to farnesyl pyrophosphate (FPP) which can be utilised for cholesterol synthesis.Accumulation of endogenous IPP can be detected by Vd2Vc9 T cells due to binding of IPP to the intracellular B30.2 domain of the transmembrane protein BTN3A1.This figure is adapted from Sanz et al., 107 and was created using Biorender.

Table 1 .
Clinical Vc9Vd2 T-cell immunotherapeutic trials with published results

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2024 The Authors.Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.

Figure 5 .
Figure 5. (a) Typical structure of simple liposomal nanocarriers which feature a phospholipid bilayer where hydrophobic drugs may be encapsulated and an aqueous core allowing for the encapsulation of hydrophilic molecules.Aminobisphosphonates (n-BPs) are hydrophilic so are typically encapsulated within the core of the nanocarrier.(b)Leaky vasculature that is common to many solid tumours allows for the enhanced permeability and retention (EPR) effect meaning that small nanoparticles can extravasate from blood vessels into tumours and will not be drained by the lymph system due to impaired lymphatic drainage.This allows nanoparticles to passively accumulate at tumour sites.The figure was created using Biorender.

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2024 The Authors.Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc. 2024 | Vol. 13 | e1492 Page 11